Polynucleotides encoding SREB1 receptor

ABSTRACT

This invention belongs to the genetic engineering field, and provides novel G protein-coupled receptor family proteins SREB1, SREB2 and SREB3 expressed in the central nervous system, genes coding for these proteins, screening methods using these proteins and so on. As one of the methods for obtaining the G protein-coupled receptor proteins of the present invention, RT-PCR is carried out using mRNA extracted from human or rat brain tissue or brain-derived cells as the template and using two primers interposing the entire portion or a part of the G protein-coupled receptor protein translation region, thereby obtaining cDNA corresponding to the G protein-coupled receptor protein or a part thereof, and the cDNA is integrated into an appropriate expression vector and expressed in a host cell.

This is a divisional of U.S. application Ser. No. 10/318,142 filed Dec.13, 2002 (now U.S. Pat. No. 6,808,899), which is a divisional of U.S.application Ser. No. 09/622,439 filed Aug. 17, 2000 (now U.S. Pat. No.6,555,344), which is a 371 of PCT/JP99/01191, filed Mar. 11, 1999.

TECHNICAL FIELD

This invention belongs to the genetic engineering field, and relates tonovel G protein-coupled receptor proteins, genes coding for these Gprotein-coupled receptor proteins, methods for producing these Gprotein-coupled receptor proteins, screening methods using these Gprotein-coupled receptor proteins, antibodies for these Gprotein-coupled receptor proteins and screening methods using theseantibodies.

BACKGROUND ART

Cell membrane receptors which transmit signals to the intracellularregion via the activation of heterotrimeric GTP binding protein aregenerally referred to as “G protein-coupled receptor”. All members ofthe G protein-coupled receptor known to date are sometimes referredgenerally to as “seven transmembrane receptor”, because they form asuper family having a common structure which has the extracellular aminoterminus and intracellular carboxyl terminus and passes through the cellmembrane seven times. The G protein-coupled receptor transmitsinformation on various physiologically active substances from cellmembranes to the intracellular region via activation of heterotrimericGTP binding protein and subsequent changes in the intracellular secondmessengers induced. As the intracellular second messengers which arecontrolled by the heterotrimeric GTP binding protein, cAMP via adenylatecyclase, Ca⁺⁺ via phospholipase C and the like are well known, and ithas been revealed recently that many cellular proteins are theirtargets, such as the control of channels and activation of proteinkinases via the heterotrimeric GTP binding protein (Gudermann, T. et al.(1997), Annu. Rev. Neurosci., 20, 399-427). The physiologically activesubstances that transmit information via the G protein-coupled receptorinclude various known physiologically active substances such asneurotransmitters, hormones, chemokine, lipid-originated signaltransducers, divalent ions and proteases. Information by thesephysiologically active substances is transmitted to the intracellularregion through their specific G protein-coupled receptor, respectively.

Several hundred types of G protein-coupled receptor have so far beencloned from eucaryote. Regarding human, hundred or more types of Gprotein-coupled receptor for corresponding endogenous ligands have beencloned and are regarded as targets of drugs for diseases. There arevarious diseases in which G protein-coupled receptor is the target, andthere exist effective drugs which act upon G protein-coupled receptor,in the respective fields of central nervous system, circulatory organsystem, inflammatory immune system, digestive organ system, motor organsystem and urinary organ/reproductive organ system (Stadel, J. et al.(1997), Trends Pharmacol. Sci., 18, 430-437). This indicates thatagonists or antagonists of G protein-coupled receptor have a highpossibility of becoming a therapeutic agent of diseases, so that studiesare being actively carried out on the discovery and identification ofnew G protein-coupled receptors.

Cloning of G protein-coupled receptor genes tends to start based ontheir structural homology in the super family in many cases, and areceptor having no correspondence to endogenous ligand is referred to as“the orphan G protein-coupled receptor”. In general, a ligand specificfor the orphan G protein-coupled receptor has not been found, so that itwas difficult to develop its agonist or antagonist. In recent years,however, it has been proposed to create a drug targeting for the orphanG protein-coupled receptor by combining the substantiated compoundlibraries and high performance high throughout screening (Stadel, J. etal. (1997), Trends Pharmacol. Sci., 18, 430-437).

That is, it is possible to screen an agonist for an orphan Gprotein-coupled receptor from a compound library by effective highthroughput system of the measurement of cAMP and Ca⁺⁺ which are secondmessengers of many G protein-coupled receptors, or the measurement ofGTPase activity and G protein binding of GTPγS which are indexes of theactivation of heterotrimeric GTP binding protein, so that it is possibleto find specific agonists and antagonists making use of such compoundsand furthermore to develop therapeutic drugs for certain diseases. Undersuch conditions, discovery of a novel G protein-coupled receptor capableof becoming a new therapeutic target of diseases is regarded as the mostimportant step in creating a medicament which acts upon Gprotein-coupled receptors.

Among G protein-coupled receptors, there is a case in which a pluralityof receptors are present for one endogenous ligand. Such receptors arereferred to as receptor family, and each receptor is called subtype.Since all of the G protein-coupled receptors have a common structurewhich passes through the cell membrane seven times, 20 to 25% of aminoacids are preserved mainly in the transmembrane region even in mutuallyindependent G protein-coupled receptors, but when they form a receptorfamily, ratio of the amino acids preserved among its subtypessignificantly increases to 35% or more, particularly to 60 to 80% amongsubtypes having high relevancy (Strader, C. D. et al. (1994), Annu. Rev.Biochem., 63, 101-132).

When development of a therapeutic drug for diseases is planned bytargeting for an endogenous ligand wherein a receptor family is present,specificity of its subtypes becomes important in many cases. This isbecause actions upon other subtype than actions upon a subtype thatmediates the main action of a drug lead to side effects in many cases.Accordingly, it is desirable to create a subtype-specific agonist orantagonist, but it is necessary to find a means for detecting thesubtype-specificity for that purpose. Currently, a method forconstructing a system in which a gene of a subtype is cloned and itsspecificity is detected using a cultured cell line or the like whichexpresses the gene is generally used.

When a novel G protein-coupled receptor is used as the target of diseasetreatment, it is highly possible that the subtype-specificity isimportant, so that discovery of a receptor family is important also inthe case of the novel G protein-coupled receptor. The homology of aminoacid sequences among independent G protein-coupled receptors is 20 to25% as a whole, but when they form a receptor family, the homologysignificantly increases in general in the family, so that it is possibleto presume whether they form a family or not, by comparing homologybetween two G protein-coupled receptors. It is possible to find novel Gprotein-coupled receptors which form a family, making use of such ameans, and when a novel G protein-coupled receptor family is discovered,it will open a way for developing a drug for disease therapy because ofthe possibility of creating a subtype-specific agonist or antagonist.

The central nervous system transmits and controls various kinds ofinformation using physiologically active substances represented byneurotransmitters. The G protein-coupled receptor is taking an importantrole in the signal transduction and control. Since many types of Gprotein-coupled receptor are present in the central nervous system, theyare used as important therapeutic targets for diseases, of the centralnervous system. For example, it is considered that the G protein-coupledreceptor of a neurotransmitter, dopamine, is a therapeutic target ofschizophrenia (Seeman, P. et al. (1997), Neuropsychopharmacology, 16,93-110), the G protein-coupled receptor of serotonin is that ofdepression (Cowen, P. J. (1991), Br. J. Psychiatry, 159 (Suppl. 12),7-14), and the G protein-coupled receptor of neuro-peptide Y is that ofeating disorder (Blomqvist, A. G. and Herzog, H. (1997), TrendsNeurosci., 20, 294-298).

It is considered that a novel G protein-coupled receptor expressing inthe central nervous system, preferably a human receptor, will lead to acandidate for a new therapeutic target of central nervous systemdiseases or to the elucidation of central nervous system functions. Inaddition, for the purpose of developing a subtype-specific drug, it isdesirable also to find a family in the case of the novel Gprotein-coupled receptor expressing in the central nervous system.Though the gene of a receptor GPR27 obtained from a mouse, having highhomology with the amino acid sequence of SREB1 which is one of the Gprotein-coupled receptors of the invention, and an amino acid sequencebased on its gene sequence have been reported (O'Dowd, B. F. et al.(1998), Genomics, 47, 310-313), no information is available to dateconcerning gene sequence and amino acid sequence of a human receptor.

DISCLOSURE OF THE INVENTION

The present invention is to provide novel G protein-coupled receptorfamily proteins expressed in the central nervous system, as the targetof therapeutic agents for central nervous system diseases.

With the aim of achieving the above object, the present inventors haveconducted intensive studies and, as a result, succeeded in isolatinggenes (SREB1, SREB2, SREB3, rSREB1, rSREB2 and rSREB3) which encodenovel G protein-coupled receptor family proteins expressed in thecentral nervous system.

Also, we have established vectors containing these genes, host cellscontaining these vectors and methods for producing these Gprotein-coupled receptor proteins using such host cells, and renderedpossible screening of these G protein-coupled receptor proteins andcompounds, peptides and antibodies capable of modifying activities ofthe G protein-coupled receptor proteins.

Illustratively, the present invention relates to

(1) a G protein-coupled receptor protein which has the amino acidsequence described in SEQ ID NOs: 2, 4, 6, 22 or 26, or a Gprotein-coupled receptor protein as an equivalent to the protein,

preferably a human origin G protein-coupled receptor protein which hasthe amino acid sequence described in SEQ ID NOs: 2, 4 or 6 or a Gprotein-coupled receptor protein as an equivalent to the protein, or arat origin G protein-coupled receptor protein which has the amino acidsequence described in SEQ ID NOs: 6, 22 or 26 or a G protein-coupledreceptor protein as an equivalent to the protein,

(2) a G protein-coupled receptor protein which has the amino acidsequence described in SEQ ID NOs: 2, 4, 6, 22 or 26,

(3) a gene which has a nucleotide sequence coding for the Gprotein-coupled receptor protein described in the item (1),

(4) a vector which contains the gene described in the item (3),

(5) a host cell which contains the vector described in the item (4),

(6) a method for producing the G protein-coupled receptor proteindescribed in the item (1) or (2), or a G protein-coupled receptorprotein as an equivalent to the protein, which comprises using the hostcell described in the item (5),

(7) a method for screening a medicament acting on the G protein-coupledreceptor protein described in the item (1) or (2), which comprisesallowing the G protein-coupled receptor protein to contact with acompound to be tested, or

(8) an antibody for the G protein-coupled receptor protein described inthe item (1) or (2) or a partial peptide thereof.

The following explains the terms to be used herein.

The term “human origin” or “rat origin” means an amino acid sequenceidentical to the amino acid sequence of a G protein-coupled receptorprotein expressing in human or rat.

The term “equivalent” of the G protein-coupled receptor protein of thepresent invention means a G protein-coupled receptor protein which isexpressed in the central nervous system and shows the same activity ofany one of the G protein-coupled receptor proteins represented by theamino acid sequences described in SEQ ID NOs: 2, 4, 6, 22 or 26.

In this connection, the G protein-coupled receptor and the Gprotein-coupled receptor protein have the same meaning.

The novel G protein-coupled receptor protein of the present invention isany one of the G protein-coupled receptor proteins represented by theamino acid sequences described in SEQ ID NOs: 2, 4, 6, 22 and 26, orequivalents thereof. Illustratively, all of G protein-coupled receptorproteins are included in the invention as long as they have the aminoacid sequence described in SEQ ID NOs: 2, 4, 6, 22 or 26, or an aminoacid sequence in which the amino acid sequence described in SEQ ID NOs:2, 4, 6, 22 or 26, has substitution, deletion or insertion of one or aplurality, preferably from 1 to 10, more preferably from 1 to 7, mostpreferably from 1 to 5, of amino acids, and have the same activity ofthe protein represented by the amino acid sequence described in SEQ IDNOs: 2, 4 or 6. Preferably, it is a human or rat origin Gprotein-coupled receptor protein having the amino acid sequencedescribed in SEQ ID NOs: 2, 4, 6, 22 or 26.

Also, the gene which has a nucleotide sequence coding for the novel Gprotein-coupled receptor protein of the invention may be any gene, aslong as it has a nucleotide sequence coding for the G protein-coupledreceptor protein represented by the amino acid sequence described in SEQID NOs: 2, 4 or 6, or an equivalent thereof. Preferably, it is a genewhich has a nucleotide sequence coding for the amino acid sequencedescribed in SEQ ID NOs: 2, 4, 6, 22 or 26. More preferably, it is agene which has a sequence of from 1 to 1,125 positions of the nucleotidesequence described in SEQ ID NO: 1, from 1 to 1,110 positions of thenucleotide sequence described in SEQ ID NO: 3, from 1 to 1,119 positionsof the nucleotide sequence described in SEQ ID NO: 5, from 1 to 1,131positions of the nucleotide sequence described in SEQ ID NO: 21, from 1to 1,110 positions of the nucleotide sequence described in SEQ ID NO: 23or from 1 to 1,119 positions of the nucleotide sequence described in SEQID NO: 25.

The gene which encodes the G protein-coupled receptor protein of theinvention can be obtained by the following methods.

1) Production Methods of Novel G Protein-coupled Receptor Protein Gene

a) First Production Method

A mRNA sample is extracted from human cells or tissue having the abilityto produce the G protein-coupled receptor protein of the invention.Next, using this mRNA as the template, two primers interposing the Gprotein-coupled receptor protein mRNA or a part of the mRNA region isprepared. The G protein-coupled receptor protein cDNA or a part thereofcan be obtained by carrying out a reverse transcriptase-polymerase chainreaction (to be referred to as RT-PCR hereinafter) suited for SREB1,SREB2 or SREB3 by modifying the conditions for denature temperature,denaturing agent addition and the like. Thereafter, the receptor proteincan be produced by integrating the thus obtained G protein-coupledreceptor cDNA or a part thereof into an appropriate expression vectorand expressing it in a host cell.

Firstly, mRNA molecules including those encoding the G protein-coupledreceptor protein of the invention are extracted by a known method fromcells or tissue, such as of the human brain or rat brain, having theability to produce the protein. Regarding the extraction method, aguanidine thiocyanate hot phenol method, a guanidinethiocyanate-guanidine hydrochloride method and the like can beexemplified, and a guanidine thiocyanate cesium chloride method can becited as a preferred method. The cells or tissue having the ability toproduce the protein can be identified by the Northern blotting methodusing a gene having a nucleotide sequence coding for the protein or apart thereof or by the Western blotting method using an antibodyspecific for the protein.

Purification of mRNA can be carried out in accordance with theconventional method, for example by adhering the mRNA to an oligo(dT)cellulose column and then eluting it therefrom. In addition, the mRNAcan be further fractionated, for example, by a sucrose density gradientcentrifugation. Alternatively, a commercially availablealready-extracted mRNA preparation may be used without carrying out themRNA extraction.

Next, a single-stranded cDNA is synthesized from the thus purified mRNAby carrying out a reverse transcriptase reaction in the presence of arandom primer or an oligo-dT primer. This synthesis can be carried outin the conventional way. The novel G protein-coupled receptor DNA ofinterest is amplified by subjecting the thus obtained single-strandedcDNA to PCR using two primers interposing a region of the gene ofinterest. The thus obtained DNA is fractionated, for example, by anagarose gel electrophoresis. As occasion demands, a DNA fragment ofinterest can be obtained by digesting the DNA with restriction enzymesand then connecting the digests.

b) Second Production Method

In addition to the above method, the gene of the invention can also beproduced making use of conventional genetic engineering techniques.Firstly, single-stranded cDNA is synthesized using the mRNA obtained bythe above method as the template and a reverse transcriptase, and thendouble-stranded cDNA is synthesized from the single-stranded cDNA.Examples of the method include the S1 nuclease method (Efstratiadis, A.et al. (1976), Cell, 7, 279-288), the Land method (Land, H. et al.(1981), Nucleic Acids Res., 9, 2251-2266), the O. Joon Yoo method (Yoo,O. J. et al. (1983), Proc. Natl. Acad. Sci. USA, 79, 1049-1053) and theOkayama-Berg method (Okayama, H. and Berg, P. (1982), Mol. Cell. Biol.,2, 161-170).

Next, the recombinant plasmid obtained by the above method is introducedinto an Escherichia coli strain, such as DH5α, to effect itstransformation, and a transformant can be selected making use oftetracycline resistance or ampicillin resistance as a marker. Forexample, when the host cell is Escherichia coli, transformation of thehost cell can be carried out by the Hanahan's method (Hanahan, D.(1983), J. Mol. Biol., 166, 557-580), namely a method in which therecombinant DNA is added to competent cells prepared in the presence ofCaCl₂ and MgCl₂ or RbCl. In this case, not only a plasmid but also alambda or the like phage vector can also be used as the vector.

A strain having DNA coding for the novel G protein-coupled receptorprotein of interest can be selected from the thus obtainedtransformants, for example by the following various methods.

(1) A Screening Method which Uses a Synthetic Oligonucleotide Probe

An oligonucleotide corresponding to the entire portion or a part of theG protein-coupled receptor protein of the invention is synthesized (inthis case, it may be either a nucleotide sequence derived using thecodon usage or a combination of plural possible nucleotide sequences,and in the latter case, their kinds can be reduced by includinginosine), this is used as a probe (labeled with ³²P or ³³P) and allowedto hybridize with DNA samples of transformants, which are denatured andfixed on a nitrocellulose filter, and then a positive strain is screenedand selected.

(2) A Screening Method which Uses a Probe Prepared by Polymerase ChainReaction

Sense primer and antisense primer oligonucleotides corresponding to apart of the G protein-coupled receptor protein of the invention aresynthesized, and polymerase chain reaction (Saiki, R. K. et al. (1988),Science, 239, 487-491) is carried out using a combination of them toeffect amplification of a DNA fragment of interest coding for the entireportion or a part of the G protein-coupled receptor protein. As thetemplate DNA to be used herein, cDNA synthesized by the reversetranscription reaction from mRNA of cells capable of producing the Gprotein-coupled receptor protein or genomic DNA can be used. The thusprepared DNA fragment is labeled with ³²P or ³³P and used as the probeto select a clone of interest by carrying out colony hybridization orplaque hybridization.

(3) A Screening Method in which the Novel G Protein-coupled ReceptorProtein is Produced in Other Animal Cells

A transformant is cultured to amplify the gene of interest, the gene istransfected into an animal cell (in this case, either a plasmid whichcan perform autonomous replication and contains a transcription promoterregion or a plasmid which can be integrated into chromosome of theanimal cell may be used) and a protein coded by the gene is produced onthe cell surface. By detecting the protein using an antibody specificfor the G protein-coupled receptor protein of the invention, a strain ofinterest having cDNA coding for the G protein-coupled receptor proteinis selected from the original transformants.

(4) A Selection Method which Uses an Antibody Specific for the GProtein-coupled Receptor Protein of the Invention

In advance, cDNA is integrated into an expression vector and protein isproduced on the surface of transformant strains, and then strainscapable of producing the G protein-coupled receptor protein are detectedusing an antibody specific for the G protein-coupled receptor protein ofthe invention and a second antibody for the first antibody, therebyselecting a strain of interest.

(5) A Method which Uses a Selective Hybridization Translation System

Samples of cDNA obtained from transformants are blotted on, for example,a nitrocellulose filter and hybridized with mRNA prepared from cellscapable of producing the G protein-coupled receptor protein of theinvention, and then the mRNA linked to the cDNA is dissociated andrecovered. The thus recovered mRNA is then translated into protein usinga protein translation system, for example by injecting into Xenopusoocyte or in a cell-free system such as a rabbit reticulocyte lysate,wheat germ or the like. A strain of interest is selected by detecting itusing an antibody for the G protein-coupled receptor protein of theinvention.

Collection of DNA which encodes the G protein-coupled receptor proteinof the invention from the thus obtained transformant of interest can becarried out in accordance with a known method (Maniatis, T. et al.(1982): “Molecular Cloning—A Laboratory Manual”, Cold Spring HarborLaboratory, NY). For example, it can be carried out by separating afraction corresponding to a plasmid DNA from cells, and cutting out acDNA region from the plasmid DNA.

c) Third Production Method

The gene which has a nucleotide sequence coding for the amino acidsequence represented by SEQ ID NOs: 2, 4, 6, 22 or 26 can also beproduced by binding DNA fragments produced by a chemical synthesismethod. Each DNA can be synthesized using a DNA synthesizer (e.g., Oligo1000M DNA Synthesizer (Beckman), 394 DNA/RNA Synthesizer (AppliedBiosystems) or the like).

d) Fourth Production Method

For the purpose of effecting expression of the function of Gprotein-coupled receptor protein of the invention by the substance thusobtained by genetic engineering techniques making use of the gene of theinvention, it is not always necessary to have all of the amino acidsequences represented by SEQ ID NOs: 2, 4, 6, 22 and 26; for example,even if it is a partial sequence or other amino acid sequence is addedthereto, such proteins are also included in the G protein-coupledreceptor protein of the invention, as long as they show the sameactivity of the G protein-coupled receptor protein represented by theamino acid sequence shown in SEQ ID NOs: 2, 4, 6, 22 or 26. Also, as isknown by the interferon gene and the like, it is considered that genesof eucaryote generally show polymorphism (e.g., see Nishi, T. et al.(1985), J. Biochem., 97, 153-159), and there is a case in which one or aplurality of amino acid are substituted by this polymorphism or a casein which the nucleotide sequence is changed but the amino acids arecompletely unchanged. In consequence, even in the case of proteins inwhich one or a plurality of amino acid residues are substituted, deletedor inserted at one or a plurality of positions in the amino acidsequence represented by SEQ ID NOs: 2, 4 or 6, it is possible that theyhave the same activity of the G protein-coupled receptor represented bythe amino acid sequence described in SEQ ID NOs: 2, 4 or 6. Theseproteins are called equivalents to the G protein-coupled receptorprotein of the invention and included in the invention. In addition, a Gprotein-coupled receptor having the rat origin amino acid sequence shownby SEQ ID NOs: 22, 24 or 26 or a G protein-coupled receptor having thesame activity of the former receptor is also included in theequivalents.

All of the genes having nucleotide sequences which encode theseequivalents to the G protein-coupled receptor protein of the inventionare included in the invention. Such various genes of the invention canalso be produced by nucleic acid chemical synthesis methods inaccordance with a usual method such as the phosphite triester method(Hunkapiller, M. et al. (1984), Nature, 10, 105-111), based on theinformation on the G protein-coupled receptor protein of the inventiondescribed in the foregoing. In this connection, codons for desired aminoacid are well known, and they can be optionally selected and determinedin the usual way, for example by taking codon usage of the host to beused into consideration (Crantham, R. et al. (1981), Nucleic Acids Res.,9, r43-r74). In addition, partial modification of codons of thesenucleotide sequences can be carried out in the usual way in accordance,for example, with the site specific mutagenesis (Mark, D. F. et al.(1984), Proc. Natl. Acad. Sci. USA, 81, 5662-5666) which uses a primercomprised of a synthetic oligonucleotide coding for the desiredmodification.

Determination of the sequence of DNA obtained by the above methods a) tod) can be carried out by, for example, the Maxam-Gilbert chemicalmodification method (Maxam, A. M. and Gilbert, E. (1980): “Methods inEnzymology”, 65, 499-559) or the dideoxy nucleotide chain terminationmethod (Messing, J. and Vieira, J. (1982), Gene, 19, 269-276) which usesM13.

Also, the vector of the invention, the host cell of the invention andthe G protein-coupled receptor protein of the invention can be obtainedby the following methods.

2) Production Method of Recombinant Protein of the G Protein-coupledReceptor of the Invention

An isolated fragment containing a gene coding for the G protein-coupledreceptor protein of the invention can transform other eucaryotic hostcell by again integrating into an appropriate vector DNA. In addition,it is possible to express the gene in respective host cells byintroducing an appropriate promoter and a sequence related to the geneexpression into these vectors.

Cells of vertebrates, insects, yeast and the like are included in theeucaryotic host cells and, though not particularly limited, examples ofcommonly used vertebrate cells include COS cell which is a simian cell(Gluzman, Y. (1981), Cell, 23, 175-182), a dihydrofolate reductasedeficient strain of Chinese hamster ovary cell (CHO) (Urlaub, G. andChasin, L. A. (1980), Proc. Natl. Acad. Sci. USA, 77, 4216-4220), humanfetal kidney HEK293 cell and 293-EBNA cell (Invitrogen) prepared byintroducing Epstein Barr virus EBNA-1 gene into the human cell.

As the expression vector for vertebrate cells, a vector which contains apromoter positioned on the upstream of the gene to be expressed, an RNAsplicing site, a polyadenylation site, transcription terminationsequence and the like can generally be used, and it may further containa replication origin as occasion demands. Examples of the expressionvector include pSV2dhfr having SV40 early promoter (Subramani, S. et al.(1981), Mol. Cell. Biol., 1, 854-864), pEF-BOS having human elongationfactor promoter (Mizushima, S. and Nagata, S. (1990), Nucleic AcidsRes., 18, 5322), pCEP4 having cytomegalovirus promoter (Invitrogen) andthe like, though not limited thereto.

In a case in which COS cell is used as the host cell, an expressionvector which has SV40 replication origin, can perform autonomous growthin COS cell and has a transcription promoter, a transcriptiontermination signal and an RNA splicing site can be used, and itsexamples include pME18S (Maruyama, K. and Takebe, Y. (1990), Med.Immunol., 20, 27-32), pEF-BOS (Mizushima, S. and Nagata, S. (1990),Nucleic Acids Res., 18, 5322), pCDM8 (Seed, B. (1987), Nature, 329,840-842) and the like. The expression vector can be incorporated intoCOS cell by, for example, the DEAE-dextran method (Luthman, H. andMagnusson, G. (1983), Nucleic Acids Res., 11, 1295-1308), the calciumphosphate-DNA co-precipitation method (Graham, F. L. and van der Ed., A.J. (1973), Virology, 52, 456-457), a method which uses FuGENE6™(Boeringer Mannheim) or the electroporation method (Neumann, E. et al.(1982), EMBO J., 1, 841-845), and a desired transformant cell can thusbe obtained.

Also, when CHO cell is used as the host cell, a transformant cellcapable of stably producing the novel G protein-coupled receptor proteincan be obtained by carrying out co-transfection of an expression vectortogether with a vector capable of expressing neo gene which functions asa G418 resistance marker, such as pRSVneo (Sambrook, J. et al. (1989):“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY) or pSV2-neo (Southern, P. J. and Berg, P. (1982), J. Mol. Appl.Genet., 1, 327-341), and selecting a G418 resistant colony. In addition,when 293-EBNA cell is used as the host cell, a desired transformant cellcan be obtained using an expression vector which has Epstein Barr virusreplication origin and can perform autonomous growth in the 293-EBNAcell, such as pCEP4 (Invitrogen).

The thus obtained desired transformant can be cultured in theconventional way, and the G protein-coupled receptor protein of theinvention is produced inside the cells or on the cell surface by thisculturing. Regarding the medium to be used in this culturing, it can beoptionally selected from various commonly used media depending on eachhost cell employed; for example, in the case of the COS cell, RPMI-1640medium, Dulbecco's modified Eagle's minimum essential medium (DMEM) orthe like can be used by adding serum components such as fetal bovineserum (FBS) and the like as occasion demands. Also, in the case of the293-EBNA cell, Dulbecco's modified Eagle's minimum essential medium(DMEM) or the like medium supplemented with serum components such asfetal bovine serum (FBS) and the like can be used by further addingG418.

The G protein-coupled receptor protein of the invention thus producedinside the cell or on the cell surface of the transformant can beseparated and purified therefrom by various known separation techniquesmaking use of physical properties, chemical properties and the like ofthe receptor protein. Illustrative examples of such techniques, to becarried out after solubilization of the receptor protein-containingmembrane fraction, include usual treatment with a protein precipitant,ultrafiltration, various liquid chromatography means such as molecularsieve chromatography (gel filtration), adsorption chromatography, ionexchange chromatography, affinity chromatography, high performanceliquid chromatography (HPLC) and the like, dialysis and combinationsthereof. In this connection, the membrane fraction can be obtained inthe usual way. For example, it can be obtained by culturing the cellswhich expressed the G protein-coupled receptor protein on the surface,suspending them in a buffer and then homogenizing and centrifuging them.Also, when the G protein-coupled receptor protein is solubilized using asolubilizing agent as mild as possible (CHAPS, Triton® X-100, digitoninor the like), characteristics of the receptor can be maintained afterthe solubilization.

By effecting expression of the G protein-coupled receptor protein of theinvention through its in-frame fusion with a marker sequence,confirmation of the expression the G protein-coupled receptor protein,confirmation of its intracellular localization, purification thereof andthe like become possible. Examples of the marker sequence include FLAG®epitope, Hexa-Histidine tag, Hemagglutinin tag, myc epitope and thelike. Also, when a specific sequence recognizable by a protease such asenterokinase, factor Xa or thrombin is inserted between a markersequence and the G protein-coupled receptor protein, the marker sequencecan be cut and removed by such a protease. For example, there is areport in which muscarinic acetylcholine receptor and Hexa-Histidine tagare connected with a thrombin-recognizing sequence (Hayashi, M. K. andHaga, T. (1996), J. Biochem., 120, 1232-1238).

A method for the screening of compounds, peptides and antibodies capableof modifying activity of the G protein-coupled receptor protein isincluded in the invention. This screening method comprises adding anagent to be tested to a system in which an index of the modification ofG protein-coupled receptor protein in response to a physiologicalcharacteristic of the G protein-coupled receptor protein is measuredmaking use of the thus constructed G protein-coupled receptor protein,and measuring the index. The following screening methods can be cited asillustrative examples of this measuring system. Also, examples of usefuldrugs to be tested include compounds or peptides which areconventionally known to have G protein-coupled receptor ligand activitybut their ability to selectively modify activity of the novel Gprotein-coupled receptor protein is not clear, known compounds andpeptides registered in chemical files but their various Gprotein-coupled receptor ligand activities are unknown, compoundsobtained by the method such as combinatorial chemistry techniques(Terrett, N. K. et al. (1995), Tetrahedron, 51, 8135-8137) and randompeptides prepared by employing a phage display (Felici, F. et al.(1991), J. Mol. Biol., 222, 301-310) or the like. In addition, culturesupernatants of microorganisms, natural components originated fromplants and marine organisms, animal tissue extracts and the like arealso objects of the screening. Also useful are compounds or peptidesobtained by chemically or biologically modifying a compound or peptideselected by the screening method of the invention.

3) Screening Methods of Ligands of the G Protein-coupled ReceptorProtein of the Invention, Namely Compounds, Peptides and Antibodieswhich Modify Activity of the G Protein-coupled Receptor Protein of theInvention

a) A Screening Method which Uses a Ligand Binding Assay Method

Compounds, peptides and antibodies which bind to the G protein-coupledreceptor protein of the invention (generally referred to as ligand) canbe screened by a ligand binding assay method. A cell membrane sampleobtained after expression of the receptor protein or a purified sampleof the receptor protein is prepared, and a ligand purified for use inthe ligand binding assay is radiation-labeled (50 to 2,000 Ci/mmol).Buffer solution, ions, pH and the like assay conditions are optimized,and the receptor protein-expressed cell membrane sample or the purifiedreceptor protein sample is incubated in the thus optimized buffer for apredetermined period of time together with the radiation-labeled ligand.After the reaction, this is filtered through, e.g., a glass filter andwashed with an appropriate amount of the buffer, and then theradioactivity remained on the filter (total binding amount) is measuredusing, e.g., a liquid scintillation counter. Nonspecific binding amountis measured by adding the unlabeled ligand in large excess in thereaction solution, and the specific binding amount is obtained bysubtracting the nonspecific binding amount from the total bindingamount. A ligand showing specific binding to the receptorprotein-expressed cell membranes or the purified receptor protein can beselected as a ligand of the G protein-coupled receptor protein of theinvention. In addition, a compound, peptide or antibody having agonistactivity, or a compound, peptide or antibody having antagonist activity,of the G protein-coupled receptor protein can be screened making use ofthe binding inhibition of the thus obtained radioactive ligand as anindex.

b) A Screening Method which Uses a GTPγS Binding Method

Compounds, peptides and antibodies capable of modifying the activity ofthe G protein-coupled receptor protein of the invention can be screenedby a GTPγS binding method (Lazareno, S. and Birdsall, N. J. M. (1993),Br. J. Pharmacol., 109, 1120-1127). Cell membranes obtained afterexpression of the receptor protein is mixed with 400 pM of GTPγS labeledwith ³⁵S in a solution of 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl₂and 50 mM GDP. After incubation in the presence or absence of an agentto be tested, this is filtered through, e.g., a glass filter and thenradioactivity of the bound GTPγS is measured using, e.g., a liquidscintillation counter. A compound, peptide or antibody having agonistactivity of the G protein-coupled receptor protein can be screenedmaking use, as an index, of the increased specific GTPγS binding in thepresence of the drug to be tested. Also, a compound, peptide or antibodyhaving antagonist activity of the G protein-coupled receptor protein canbe screened making use, as an index, of the suppression of increase inthe GTPγS binding by the thus obtained compound, peptide or antibodyhaving agonist activity.

c) A Screening Method which Uses Changes in the Intracellular Ca⁺⁺ andcAMP Concentrations

Many G protein-coupled receptor proteins induce increase in Ca⁺⁺ and/orincrease or decrease in cAMP concentration in the cells caused by anagonist stimulus. Accordingly, compounds, peptides and antibodiescapable of modifying the activity of the G protein-coupled receptorprotein of the invention can be screened making use of the changes inthe intracellular Ca⁺⁺ or cAMP concentration. The Ca⁺⁺ concentration ismeasured using fura2 and the like, and the cAMP concentration ismeasured using a commercially available cAMP assay kit (by Amersham,etc.).

Alternatively, it is possible to measure the Ca⁺⁺ and cAMPconcentrations indirectly, by detecting the transcription activity of agene whose transcription amount is controlled depending on the Ca⁺⁺ andcAMP concentrations. A sample such as a compound, a peptide, a tissueextract or the like is allowed to react for a predetermined period oftime with cells in which the receptor protein is expressed or host cellsin which the receptor protein is not expressed (control cells), and theCa⁺⁺ and cAMP concentrations are measured directly or indirectly. Acompound, peptide or antibody having agonist activity can be screenedmaking use, as an index, of the increase in Ca⁺⁺ and/or increase ordecrease in cAMP concentration in the receptor protein-expressed cellsby comparing with the control cells. Also, a compound, peptide orantibody having antagonist activity of the G protein-coupled receptorprotein can be screened making use, as an index, of the increase in Ca⁺⁺and/or increase or decrease in cAMP concentration caused by the thusobtained compound, peptide or antibody having agonist activity.

d) A Screening Method which Uses Microphysiometer

Upon various signal responses of cells, trace amount of hydrogen ionsoutflow into the extracellular moiety is detected. Most of this outflowof hydrogen ions occur when metabolites formed by the fuel consumptionof cells to obtain energy for their responses are increased or whensignals of the cells are transmitted directly to the hydrogen ion pump.Since the G protein-coupled receptor protein of the invention requiresenergy for its signal transmission, outflow of hydrogen ions occurs whenthe receptor is activated. Since changes in pH caused by such a traceoutflow of hydrogen ions in a medium around cells can be detected byCYTOSENSOR® Microphysiometer (Molecular Devices), it can be used for thedetection of the activation energy consuming receptors.

A compound, a peptide, a tissue extract or the like is allowed to reactfor a predetermined period of time with cells in which the receptorprotein is expressed or host cells in which the receptor protein is notexpressed (control cells), and changes in the pH due to outflow ofhydrogen ions are measured. A compound, peptide or antibody havingagonist activity can be screened making use, as an index, of the changesin pH caused by the outflow of hydrogen ions from the receptorprotein-expressed cells by comparing with the control cells. Also, acompound, peptide or antibody having antagonist activity of the Gprotein-coupled receptor protein can be screened making use, as anindex, of the changes in pH due to the outflow of hydrogen ions causedby the thus obtained compound, peptide or antibody having agonistactivity.

A medicament which contains as the active ingredient a compound, peptideor antibody capable of significantly modifying the activity of the Gprotein-coupled receptor protein or a G protein-coupled receptor proteinselected by the screening method is included in the invention.

The antibody, such as a polyclonal antibody or monoclonal antibody,which reacts with the G protein-coupled receptor protein of theinvention can be obtained by directly administering the novel Gprotein-coupled receptor protein or a fragment of the G protein-coupledreceptor protein to various animals. It can also be obtained by a DNAvaccine method (Raz, E. et al. (1994), Proc. Natl. Acad. Sci. USA, 91,9519-9523; Donnelly, J. J. et al. (1996), J. Infect. Dis., 173, 314-320)using a plasmid in which a gene which encodes the G protein-coupledreceptor protein of the invention is introduced.

The polyclonal antibody is produced from sera or eggs of an animal(e.g., rabbit, rat, goat, fowl or the like) immunized and sensitized byemulsifying the G protein-coupled receptor protein or a fragment thereofin an appropriate adjuvant such as complete Freund's adjuvant andadministering it by intraperitoneal, subcutaneous or intravenousinjection. The polyclonal antibody thus produced from sera or eggs canbe separated and purified by the usual protein isolation purificationmethods. Examples of such methods include centrifugation, dialysis,salting out with ammonium sulfate, and chromatographic techniques usingcarriers such as DEAE-cellulose, hydroxyapatite, protein A agarose andthe like.

An active antibody fragment containing a part of the antibody, such asF(ab′)2, Fab, Fab′ or Fv, can be obtained by digesting the thusseparated and purified antibody with a proteolytic enzyme such aspepsin, papain or the like in the usual way and subsequently separatingand purifying it by the usual protein isolation purification methods.

It is possible for those skilled in the art to easily produce amonoclonal antibody by the cell fusion method of Kohler and Milstein(Kohler, G. and Milstein, C. (1975), Nature, 256, 495-497).

Mice are immunized by intraperitoneal, subcutaneous or intravenousinoculation of an emulsion prepared by emulsifying the G protein-coupledreceptor protein of the invention or a fragment thereof in anappropriate adjuvant such as complete Freund's adjuvant, several timesrepeatedly at intervals of several weeks. After final immunization,spleen cells are collected and fused with myeloma cells to prepare ahybridoma.

Myeloma cells having hypoxanthine-guanine phosphoribosyltransferasedeficiency, thymidine kinase deficiency or the like marker, such asmouse myeloma cell strain P3X63Ag8. U1, are used as the myeloma cellsfor obtaining the hybridoma. Also, polyethylene glycol is used as thefusing agent. In addition, Eagle's minimum essential medium, Dulbecco'smodified minimum essential medium, RPMI-1640 or the like generally usedmedium is optionally supplemented with 10 to 30% of fetal bovine serumand used as the medium for the preparation of the hybridoma. Fusedstrains are selected by the HAT selection method. Screening of hybridomais carried out using a conventional method such as the culturesupernatant by ELISA, immunohistological staining or the like or by thescreening method described in the foregoing, and a hybridoma clonesecreting the antibody of interest is selected. Also, monoclonal natureof the hybridoma is confirmed by repeating subcloning by means oflimiting dilution analysis. When the thus obtained hybridoma is culturedfor 2 to 4 days in a medium or for 10 to 20 days in the abdominal cavityof a BALB/c mice pretreated with pristane, the antibody in an amountsufficient for purification is produced.

The thus produced monoclonal antibody can be separated and purified fromthe culture supernatant or ascites by the usual protein isolationpurification methods. Examples of such methods include centrifugation,dialysis, salting out with ammonium sulfate, and chromatographictechniques using carriers such as DEAE-cellulose, hydroxyapatite,protein A agarose and the like. In addition, the monoclonal antibody oran antibody fragment containing a part thereof can also be produced byintegrating entire portion or a part of a gene coding for the antibodyinto an expression vector and introducing into Escherichia coli, yeastor animal cells. An active antibody fragment containing a part of theantibody, such as F(ab′)2, Fab, Fab′ or Fv, can be obtained by digestingthe thus separated and purified antibody with a proteolytic enzyme suchas pepsin, papain or the like in the usual way and subsequentlyseparating and purifying it by the usual protein isolation purificationmethods.

In addition, it is possible to obtain an antibody capable of reactingwith the G protein-coupled receptor protein of the invention as singlechain Fv or Fab by the method of Clackson et al. or Zebedee et al.(Clackson, T. et al. (1991), Nature, 352, 624-628; Zebedee, S. et al.(1992), Proc. Natl. Acad. Sci. USA, 89, 3175-3179). It is also possibleto obtain a human antibody by immunizing a transgenic mouse in which amouse antibody gene is replaced by a human antibody gene (Lonberg, N. etal. (1994), Nature, 368, 856-859).

The medicament of the invention is characterized in that it has a novelpharmacological action to selectively control activity of the Gprotein-coupled receptor, and examples of the use of the medicamentinclude central nervous system diseases which are induced byabnormalities of the G protein-coupled receptor activity (acceleration,reduction, denaturation and the like) or which express the abnormalitiesas complications.

The pharmaceutical preparation which contains a compound, peptide,antibody or antibody fragment capable of modifying activity of the Gprotein-coupled receptor protein of the invention, as the activeingredient, can be prepared using carriers, fillers and other additivesgenerally used in the preparation of medicaments, in response to eachtype of the active ingredient.

Examples of its administration include oral administration in the formof tablets, pills, capsules, granules, fine granules, powders, oralsolutions and the like, and parenteral administration in the form ofintravenous, intramuscular and the like injections, suppositories,percutaneous preparations, transmucosal absorption preparations and thelike. Particularly, in the case of peptides which are digested in thestomach, intravenous injection or the like parenteral administration isdesirable.

In the solid composition for use in the oral administration according tothe present invention, one or more active substances are mixed with atleast one inert diluent such as lactose, mannitol, glucose,microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolidone or aluminum magnesium metasilicate. In the usual way, thecomposition may contain additives other than the inert diluent, forexample, a lubricant, a disintegrating agent, a stabilizing agent and asolubilizing or solubilization assisting agent. If necessary, tablets orpills may be coated with a sugar coating or a film of a gastric orenteric substance.

The liquid composition for oral administration includes emulsions,solutions, suspensions, syrups and elixirs and contains a generally usedinert diluent such as purified water or ethanol. In addition to theinert diluent, this composition may also contain other additives such asmoistening agents, suspending agents, sweeteners, flavors andantiseptics.

The injections for parenteral administration includes aseptic aqueous ornon-aqueous solutions, suspensions and emulsions. Examples of thediluent for use in the aqueous solutions and suspensions includedistilled water for injection use and physiological saline. Examples ofthe diluent for use in the non-aqueous solutions and suspensions includepropylene glycol, polyethylene glycol, plant oils (e.g., olive oil),alcohols (e.g., ethanol), polysorbate 80 and the like. Such acomposition may further contain a moistening agent, an emulsifyingagent, a dispersing agent, a stabilizing agent, a solubilizing orsolubilization assisting agent, an antiseptic and the like. Thesecompositions are sterilized for example by filtration through a bacteriaretaining filter, blending of a germicide or irradiation. Alternatively,they may be used by firstly making into sterile solid compositions anddissolving them in sterile water or other sterile solvent for injectionuse prior to their use.

The dose is optionally decided by taking into consideration strength ofeach active ingredient selected by the screening method described in theforegoing and symptoms, age, sex and the like of each patient to beadministered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows alignment of amino acid sequences of SREB1, SREB2 andSREB3.

FIG. 2 shows a result of Northern analysis of SREB1 in human organs.

FIG. 3 shows a result of Northern analysis of SREB1 in each region ofhuman brain.

FIG. 4 shows a result of Northern analysis of SREB2 in human organs.

FIG. 5 shows a result of Northern analysis of SREB2 in each region ofhuman brain.

FIG. 6 shows a result of Northern analysis of SREB3 in human organs.

FIG. 7 shows a result of Northern analysis of SREB3 in each region ofhuman brain.

FIG. 8 shows a result confirming expression of SREB1, SREB2 or SREB3protein.

FIG. 9 shows binding activity of anti-3LO antibody for SREB1, SREB2 orSREB3.

FIG. 10 shows binding activity of anti-C24 antibody for SREB1.

FIG. 11 shows pCRE-Luc derived luciferase activity in cells in whichSREB1, SREB2 or SREB3 was introduced.

FIG. 12 shows pSRE-Luc derived luciferase activity in cells in whichSREB1, SREB2 or SREB3 was introduced.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to disclose the invention further illustratively, Examples aredescribed in the following, but the invention is not limited to theseExamples. In this connection, unless otherwise noted, they can becarried out in accordance with known methods (Maniatis, T. et al.(1982): “Molecular Cloning—A Laboratory Manual”, Cold Spring HarborLaboratory, NY).

EXAMPLE 1 Isolation of Genes Coding for the Novel G Protein-coupledReceptor Family Proteins

Full length cDNA coding for the G protein-coupled receptor familyprotein (SREB1, SREB2 or SREB3) of the invention was obtained by RT-PCRusing human brain origin poly A⁺ RNA (Clontech) as the template.

In the amplification of the novel G protein-coupled receptor humanSREB1, 5′-AAAATCTAGA CGCGATGGCGAACGCGAGCGA-3′ (SEQ ID NO: 7) was used asthe forward primer, and 5′-AAAATCTAGA GTCTATGTGGCGGGGCCTCCC-3′ (SEQ IDNO: 8) as the reverse primer (XbaI site is added to each 5′ terminus).RT-PCR was carried out using Pfu DNA polymerase (Stratagene) and byrepeating a cycle of 98° C. (20 seconds)/64° C. (30 seconds)/74° C. (3minutes) 34 times in the presence of 5% formamide. As the result, a DNAfragment of about 1.2 kbp was amplified. This fragment was digested withXbaI and then cloned using pCEP4 plasmid (Invitrogen). Since the pCEP4plasmid contains CMV promoter which shows strong promoter activity inanimal cells, it can be used in expressing recombinant proteins inanimal cells. Nucleotide sequence of the thus obtained clone wasanalyzed by the dideoxy terminator method using ABI377 DNA Sequencer(Applied Biosystems). The thus revealed sequence is shown in SEQ ID NO:1 of the Sequence Listing.

This sequence contains an open reading frame of 1,125 bases (from the1st position to the 1125th position of SEQ ID NO: 1). An amino acidsequence (375 amino acids) deduced from the open reading frame is shownin SEQ ID NO: 2 of the Sequence Listing. Since the deduced amino acidsequence contains seven hydrophobic regions considered to be thetransmembrane domains which is a characteristic of the G protein-coupledreceptor, it was found that this gene encodes the G protein-coupledreceptor.

In the amplification of the novel G protein-coupled receptor humanSREB2, 5′-AAAATCTAGA TCTATGGCGAACTATAGCCATGCA-3′ (SEQ ID NO: 9) was usedas the forward primer, and 5′-AAAATCTAGA AAGGCTAAAGATTTACAGATGCTCC-3′(SEQ ID NO: 10) as the reverse primer (XbaI site is added to each 5′terminus). RT-PCR was carried out using Pfu DNA polymerase (Stratagene)and by repeating a cycle of 96° C. (20 seconds)/54° C. (30 seconds)/74°C. (3 minutes) 34 times. As the result, a DNA fragment of about 1.2 kbpwas amplified. This fragment was digested with XbaI and then clonedusing pCEP4 plasmid (Invitrogen). Nucleotide sequence of the thusobtained clone was analyzed by the dideoxy terminator method usingABI377 DNA Sequencer (Applied Biosystems). The thus revealed sequence isshown in SEQ ID NO: 3 of the Sequence Listing.

This sequence contains an open reading frame of 1,110 bases (from the1st position to the 1110th position of SEQ ID NO: 3). An amino acidsequence (370 amino acids) deduced from the open reading frame is shownin SEQ ID NO: 4 of the Sequence Listing. Since the deduced amino acidsequence contains seven hydrophobic regions considered to be thetransmembrane domains which is a characteristic of the G protein-coupledreceptor, it was found that this gene encodes the G protein-coupledreceptor.

In the amplification of the novel G protein-coupled receptor humanSREB3, 5′-AAAATCTAGA GTATGGCCAACACTACCGGAGAG-3′ (SEQ ID NO: 11) was usedas the forward primer, and 5′-AAAATCTAGA CCTGTCTGCCTACCAGCCTGC-3′ (SEQID NO: 12) as the reverse primer (XbaI site is added to each 5′terminus). RT-PCR was carried out using Pfu DNA polymerase (Stratagene)and by repeating a cycle of 98° C. (20 seconds)/62° C. (30 seconds)/74°C. (3 minutes) 34 times in the presence of 5% formamide. As the result,a DNA fragment of about 1.2 kbp was amplified. This fragment wasdigested with XbaI and then cloned using pCEP4 plasmid (Invitrogen).Nucleotide sequence of the thus obtained clone was analyzed by thedideoxy terminator method using ABI377 DNA Sequencer (AppliedBiosystems). The thus revealed sequence is shown in SEQ ID NO: 5 of theSequence Listing.

This sequence contains an open reading frame of 1,119 bases (from the1st position to the 1119th position of SEQ ID NO: 5). An amino acidsequence (373 amino acids) deduced from the open reading frame is shownin SEQ ID NO: 6 of the Sequence Listing. Since the deduced amino acidsequence contains seven hydrophobic regions considered to be thetransmembrane domains which is a characteristic of the G protein-coupledreceptor, it was found that this gene encodes the G protein-coupledreceptor.

Homology of the novel G protein-coupled receptor SREB family (SREB1,SREB2 or SREB3) with a known G protein-coupled receptor family is 25% orless, respectively.

On the other hand, homology of SREB1 with SREB2 is 52%, homology ofSREB1 with SREB3 is 52% and homology of SREB2 with SREB3 is 63%, whichare significantly higher than the homology with known G protein-coupledreceptors (FIG. 1). This fact shows that the G protein-coupled receptorsSREB1, SREB2 and SREB3 of the invention form a novel G protein-coupledreceptor family independent of the known G protein-coupled receptors.

EXAMPLE 2 Expression Distribution of Human Novel G Protein-coupledReceptor Family Genes in Tissues

Expression distribution of the G protein-coupled receptor genes of theinvention was analyzed by the northern blot hybridization method. A cDNAfragment (from the 722nd position to the 1054th position in SEQ IDNO: 1) was used as the probe of human SREB1. Poly A⁺ RNA (2 μg)originated from each of human organs was blotted on a membrane(Clontech), and its hybridization with the probe was carried out at 42°C. (18 hours) in a solution containing 50% formamide, 5×SSPE, 10×Denhardt's solution, 2% SDS and 100 μg/ml denatured salmon sperm DNA.The membrane was finally washed twice (65° C. for 30 minutes) with asolution containing 0.2×SSC and 0.1% SDS. As shown in FIG. 2, when thenorthern analysis was carried out on each of human organs (heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, large intestine andperipheral leukocyte), 3 kb of mRNA was detected in the brain, ovary,testis, heart and prostate, and 3 kb and 2.3 kb mRNA in the peripheralleukocyte. Also, a signal of 3 kb was slightly detected in the pancreas,too. In addition, the northern analysis was also carried out on each ofthe regions of the human brain (amygdala, caudate nucleus, corpuscallosum, hippocampus, substania nigra, subthalamic nucleus, thalamus,cerebellum, cerebral cortex, medulla, spinal cord, occipital lobe,frontal lobe, temporal lobe and putamen). Since the 3 kb mRNA of the Gprotein-coupled receptor human SREB1 gene of the invention was detectedin all of the examined human brain regions, it was found that it isexpressed broadly in the human brain (FIG. 3).

A cDNA fragment (from the 558th position to the 888th position in SEQ IDNO: 3) was used as the probe of human SREB2. When the northern analysiswas carried out under the same conditions, 3.2 kb mRNA was detected inthe brain, and 2.4 kb, 3.5 kb and 6.3 kb mRNA in the testis, as shown inFIG. 4. Also, the signal of 3.5 kb was detected in the placenta andspleen, and the signal of 3.2 kb in small intestine, all slightly. Amongregions in the brain, the 3.2 kb mRNA of the G protein-coupled receptorhuman SREB2 gene of the invention was abundantly detected in theamygdala, caudate nucleus, hippocampus, substania nigra, subthalamicnucleus, thalamus, cerebellum, cerebral cortexes and putamen, but not somuch in the corpus callosum, medulla and spinal cord. In addition, asignal of 7.8 kb was slightly detected in each of the grain regions(FIG. 5)

A cDNA fragment (from the 1st position to the 652nd position in SEQ IDNO: 5) was used as the probe of human SREB3. When the northern analysiswas carried out under the same conditions, 4 kb and 5.1 kb mRNA wasdetected in the brain, and 4 kb, 5.1 kb and 9.7 kb mRNA in the ovary, asshown in FIG. 6. The G protein-coupled receptor human SREB3 gene of theinvention was detected in each region of the brain as signals of mainly4 kb, 5.1 kb and slightly 9.7 kb, and the 4 kb mRNA was detected in theamygdala, hippocampus, subthalamic nucleus, cerebellum and cerebralcortex, and the 5.1 kb mRNA in the substania nigra, subthalamic nucleusand spinal cord, relatively abundantly (FIG. 7).

The above results showed that the G protein-coupled receptor familygenes SREB1, SREB2 and SREB3 of the invention are expressed mainly inthe central nervous system and urinary organ/reproductive organ system.

EXAMPLE 3 Confirmation of the Expression of the Novel Human GProtein-coupled Receptor Family Proteins

pCEP4 (Invitrogen) was used as the expression vector for expressinghuman SREB1, SREB2 or SREB3. In this case, in order to fuse a FLAG®epitope as a marker sequence with the N terminus of human SREB1, SREB2or SREB3, ATGGACTACAAGGACGACGATGACAAGGGGATCCTG (SEQ ID NO: 13) wasinserted into the 5′ terminus of the protein coding sequence of SREB1,SREB2 or SREB3. The thus constructed plasmids were named pCEP4-FL-SREB1,pCEP4-FL-SREB2 and pCEP4-FL-SREB3, respectively. By the use of theseplasmids, a polypeptide in which a sequence Met Asp Tyr Lys Asp Asp AspAsp Lys Gly Ile Leu (SEQ ID NO: 14) is fused with the N terminus of thepolypeptide of SREB1, SREB2 or SREB3 is expressed.

A 1×10⁶ cells portion of 293-EBNA (Invitrogen) was inoculated into a 10cm Petri dish and cultured for 1 day, and then gene transfer of 8 μg ofpCEP4-FL-SREB1, pCEP4-FL-SREB2, pCEP4-FL-SREB3 or pCEP4-FL (vectoralone) was carried out using FuGENE6™ (Boeringer Mannheim). After thegene transfer, the cells were cultured for 1 day, harvested, washed,suspended in 20 mM of Tris-HCl (pH 7.4)/150 mM NaCl/Complete™ (BoeringerMannheim) and then homogenized using Polytron. The homogenate was mixedwith Triton® X-100, Digitonin and sodium cholate to final concentrationsof 0.2%, 0.1% and 0.2% and then solubilized by incubating at 4° C. for 2hours. Immunoprecipitation of the FLAG® epitope fusion protein from thethus solubilized sample was effected using M2-agarose (Sigma). Theimmune precipitate was eluted with 200 μM FLAG® peptide/20 mM Tris-HCl(pH 7.4)/150 mM NaCl. The eluted sample was concentrated, subjected toelectrophoresis using SDS/10%-20% acrylamide gel (Daiichi PureChemicals) and then transferred on a PVDF membrane using a blottingapparatus. The PVDF membrane after the transfer was subjected toblocking and then allowed to react with a mouse anti-FLAG® monoclonalantibody (M2; Sigma) and a horseradish peroxidase-labeled rabbitanti-mouse IgG polyclonal antibody (Zymed) in that order. After thereaction, expression of SREB1, SREB2 or SREB3 protein was confirmedusing ECL™ Western Blotting Detection System (Amersham-Pharmacia) (FIG.8).

The protein capable of reacting with the anti-FLAG® antibody was notpresent in the cells in which pCEP4-FL was introduced but detected inthe cells in which pCEP4-FL-SREB1, pCEP4-FL-SREB2 or pCEP4-FL-SREB3 wasintroduced as a band of 35 to 45 kDa. Estimated molecular weights ofhuman SREB1, human SREB2 and human SREB3 were 39.8 kDa, 42.0 kDa and41.5 kDa, respectively, and their bands were found at positions ofalmost expected molecular weights. In addition, a band of 65 to 75 kDaconsidered to be a dimer was detected in the case of human SREB1.

EXAMPLE 4 Isolation of Gene Coding for Rat SREB1 (rSREB1), Rat SREB2(rSREB2) or Rat SREB3 (rSREB3) Protein

Complete length cDNA coding for rSREB2, rSREB2 or rSREB3 was obtained byRT-PCR using rat brain origin poly A⁺ RNA (Clontech) as the template.

In the amplification of rSREB1, 5′-AAAATCTAGACGGCGATGGCGAACGCTAGTGA-3′(SEQ ID NO: 15) was used as the forward primer, and 5′-AAAATCTAGACACTTTGAGAGTCTTGTGAAGGC-3′ (SEQ ID NO: 16) as the reverse primer (XbaIsite is added to each 5′ terminus). Amplification, cloning andnucleotide sequence determination of cDNA were carried out by the samemethods of Example 1. The thus revealed sequence is shown in SEQ ID NO:21 of the Sequence Listing.

This sequence contains an open reading frame of 1,131 bases (from the1st position to the 1131st position of SEQ ID NO: 21). An amino acidsequence (377 amino acids) deduced from the open reading frame is shownin SEQ ID NO: 22 of the Sequence Listing. Since the deduced amino acidsequence coincided in 97% frequency with the human SREB1, it was foundthat this gene encodes rSREB1.

In the amplification of rSREB2, 5′-AAAATCTAGATCTATGGCGAACTATAGCCATGC-3′(SEQ ID NO: 17) was used as the forward primer, and 5′-AAAATCTAGAAAGGCTAAAGATTTACAGATGCTCC-3′ (SEQ ID NO: 18) as the reverse primer (XbaIsite is added to each 5′ terminus). Amplification, cloning andnucleotide sequence determination of cDNA were carried out by the samemethods of Example 1. The thus revealed sequence is shown in SEQ ID NO:23 of the Sequence Listing.

This sequence contains an open reading frame of 1,110 bases (from the1st position to the 1110th position of SEQ ID NO: 23). An amino acidsequence (370 amino acids) deduced from the open reading frame is shownin SEQ ID NO: 24 of the Sequence Listing. Since the deduced amino acidsequence coincided in 100% frequency with the human SREB2, it was foundthat this gene encodes rSREB2.

In the amplification of rSREB3, 5′-AAAATCTAGACAAATACTGAACTGGCCGATCCCC-3′(SEQ ID NO: 19) was used as the forward primer, and 5′-AAAATCTAGATGTTGGCCCCAGTATGGTGATCAT-3′ (SEQ ID NO: 20) as the reverse primer (XbaIsite is added to each 5′ terminus). Amplification, cloning andnucleotide sequence determination of cDNA were carried out by the samemethods of Example 1. The thus revealed sequence is shown in SEQ ID NO:25 of the Sequence Listing.

This sequence contains an open reading frame of 1,119 bases (from the1st position to the 1119th position of SEQ ID NO: 25). An amino acidsequence (373 amino acids) deduced from the open reading frame is shownin SEQ ID NO: 26 of the Sequence Listing. Since the deduced amino acidsequence coincided in 99% frequency with the human SREB3, it was foundthat this gene encodes rSREB3.

EXAMPLE 5 Preparation of Antibody for Human SREB1

A partial amino acid sequence of human SREB1 was fused withglutathione-S-transferase (GST) and used as the immunization antigen forthe preparation of antibody for human SREB1. Illustratively, a cDNAfragment corresponding to a region of from the 208th position to the282nd position (3LO) and a region of from the 351st position to the375th position (C24) of the human SREB1 amino acid sequence (SEQ ID NO:2) was amplified by PCR in an way to bind cleavage sites of restrictionenzymes BamHI and XhoI, and inserted between BamHI and XhoI sites of GSTGene Fusion Vector (pGEX-5X-1: Amersham-Pharmacia). Competent cells ofan Escherichia coli strain BL21(DE3)pLysS (Novagen) were transformedwith the thus constructed plasmid. By culturing the transformant andinducing expression of the gene with 1 mM IPTG, a GST-3LO fusion proteinand a GST-C24 fusion protein were expressed in the E. coli cells. TheGST-3LO and GST-C24 were purified from disrupted E. coli cells usingGlutathione Sepharose 4B (Amersham-Pharmacia) in accordance with theinstruction attached thereto.

The thus purified GST-3LO fusion protein was mixed with the same amountof Freund's complete adjuvant (CalBioChem) and emulsified, and theemulsion was administered to a female white Leghorn (140 days of age) ataround the bursa of Fabricius. The initial dose was 1 mg, and it wasadministered thereafter in 0.5 mg portions 4 times at intervals of 2weeks. After the final immunization, eggs were collected, the yolk ofeggs was diluted with physiological saline and defatted using dextransulfate, and then IgY was purified using DEAE Sepharose®(Amersham-Pharmacia) to obtain anti-3LO antibody. Also, the purifiedGST-C24 fusion protein was mixed with the same amount of TiterMax® Gold(CytRX) and emulsified, and the emulsion was administered under thedorsal skin of a Japanese white rabbit (6 weeks of age). Its initialdose was 1 mg, and it was administered thereafter in 0.5 mg portions 2times at intervals of 2 weeks. After the final immunization, blood wascollected, and IgG was purified from the serum using Protein GSepharose® (Amersham-Pharmacia) in accordance with the instructionattached thereto, thereby obtaining anti-C24 antibody.

Since the anti-3LO antibody uses a region of from the 208th position tothe 282nd position of the human SREB1 amino acid sequence (SEQ ID NO: 2)as the antigen and this partial amino acid sequence contains a largenumber of sequences common to SREB1, SREB2 and SREB3 (cf. FIG. 1), thereis a possibility that the anti-3LO antibody commonly recognizes SREB1,SREB2 and SREB3. Also, since the anti-C24 antibody uses a region of fromthe 351st position to the 375th position of the human SREB1 amino acidsequence (SEQ ID NO: 2) as the antigen and this partial amino acidsequence is a sequence in which SREB2 and 3 are not present but SREB1alone is present (cf. FIG. 1), there is a possibility that the anti-C24antibody recognizes only SREB1. In consequence, in order to confirm thespecificity of anti-3LO antibody and anti-C24 antibody, Western blottingwas carried out using the immune precipitate of anti-FLAG® antibody of293-EBNA in which SREB1, SREB2 or SREB3 was expressed, prepared inExample 3, and the anti-3LO antibody and anti-C24 antibody.

Illustratively, each sample was subjected to electrophoresis usingSDS/10%-20% acrylamide gel (Daiichi Pure Chemicals) and then transferredon a PVDF membrane using a blotting apparatus. The PVDF membrane afterthe transfer was subjected to blocking and then allowed to react with 10μg/ml of the anti-3LO antibody and a horseradish peroxidase-labeledrabbit anti-chicken IgG polyclonal antibody (Zymed) in that order orwith 10 μg/ml of the anti-C24 antibody and a horseradishperoxidase-labeled goat anti-rabbit IgG polyclonal antibody (MBL) inthat order. After the reaction, color development was carried out usingECL™ Western Blotting Detection System (Amersham-Pharmacia). A bandreacting with the anti-3LO antibody was detected at the same position ofthe anti-FLAG® antibody of Example 3 in cells in which pCEP4-FL-SREB1,pCEP4-FL-SREB2 or pCEP4-FL-SREB3 (FIG. 9) was introduced. Also, a bandreacting with the anti-C24 antibody was detected at the same position ofthe anti-FLAG® antibody of Example 3 only in the cells in whichpCEP4-FL-SREB1 was introduced (FIG. 10).

Based on the above results, it was confirmed that the anti-3LO antibodyis an antibody which recognizes SREB1, SREB2 or SREB3, and the anti-C24antibody is an antibody which recognizes only SREB1. The use of theseantibodies has rendered possible the detection of natural SREB1, SREB2or SREB3 by the method such as the Western blotting, immunohistologicalstaining or the like.

EXAMPLE 6 Evaluation of Transcription Activity via cAMP-response Element(CRE) or Serum Response Element (SRE) in Human SREB1-, SREB2- orSREB3-introduced Cells

Increase in the transcription activity mediated by CRE or SRE is inducedby the activation of the intracellular information transmission systemof various G protein-coupled receptors (Lolait, S. J. et al. (1992),Nature, 357, 336-339; Hoeltzel, W. L. et al. (1997), Am. J. Physiol.,273, C2037-C2045; An, S. et al. (1998), J. Biol. Chem., 273, 7906-7910).Also, it is known that the intracellular information transmission systemof G protein-coupled receptors is partially activated via a certaintransitional active conformation even in the absence of agonist(Kenakin, T. (1995), Trens. Pharmacol. Sci., 16, 188-192). Accordingly,if changes in the CRE- or SRE-mediated transcription activity in SREB1-,SREB2- or SREB3-introduced cells are found even in the absence ofagonist, it can be confirmed that the G protein-coupled receptor isfunctional and that activation of the G protein-coupled receptorintracellular information transmission system leads to the CRE- andSRE-mediated transcription activity.

Using pEF-BOS (Mizushima, S. and Nagata, S. (1990), Nucleic Acids Res.,18, 5322) as the expression vector for expressing human SREB1, SREB2 orSREB3, pEF-BOS-SREB1, pEF-BOS-SREB2 and pEF-BOS-SREB3 were prepared. A8×10⁴ cells portion of 293-EBNA (Invitrogen) was inoculated into a24-well plate and cultured for 1 day, and then gene transfer of 250 ngof pEF-BOS-SREB1, pEF-BOS-SREB2, pEF-BOS-SREB3 or pEF-BOS (vector alone)was carried out together with 25 ng of a CRE-reporter plasmid pCRE-Luc(Stratagene) or an SRE-reporter plasmid pSRE-Luc (Stratagene), usingFuGENE6™ (Boeringer Mannheim) (3 wells for each). After the genetransfer, the cells were lysed at every 12 hours using PicaGene® CellCulture Lysis Reagent Luc (Nippon Gene), and the activity of luciferaseproduced from each reporter plasmid was measured using PicaGene®Luminescence Kit (Nippon Gene).

The luciferase activity in the SREB1-, SREB2- or SREB3-introduced cellsafter 24 hours of the gene transfer was treated as a relative activityto the luciferase activity of the vector alone introduced cells(control) (the control was defined as 1), with the results shown in FIG.11 (pCRE-Luc derived luciferase activity) and FIG. 12 (pSRE-Luc derivedluciferase activity). The CRE-mediated transcription activity increasedmost sharply in the SREB1-introduced cells and also increasedsignificantly in the SREB2- and SREB3-introduced cells in comparisonwith the control. On the other hand, the SRE-mediated transcriptionactivity increased most sharply in the SREB2-introduced cells and alsoincreased significantly in the SREB1- and SREB3-introduced cells incomparison with the control.

It was revealed by these results that the SREB1, SREB2 and SREB3 arefunctional receptors, and activation of the intracellular informationtransmission system of these G protein-coupled receptors leads to theincrease in the CRE- or SRE-mediated transcription activity.

INDUSTRIAL APPLICABILITY

Novel G protein-coupled receptor family proteins SREB1, SREB2 and SREB3expressing in the central nervous system, genes coding for theseproteins, vectors containing these genes, host cells containing thesevectors and methods for producing these G protein-coupled receptorproteins were provided by the present invention.

Also, it rendered possible to screen new medicaments, particularly newtherapeutic agents for central nervous system diseases, through thescreening of compounds, peptides and antibodies capable of modifyingactivities of the G protein-coupled receptor proteins of the inventionby allowing the G protein-coupled receptors to contact with drugs to betested.

Regarding the medicament of the invention which contains, as the activeingredient, a compound, peptide or antibody capable of specificallymodifying activity of the G protein-coupled receptor proteins expressingin the central nervous system, its usefulness as therapeutic agents andthe like for functional/organic diseases of the central nervous system.Also, since the G protein-coupled receptor family proteins of theinvention are expressed not only in the central nervous system but alsoin the urinary organ/reproductive organ system, usefulness astherapeutic drugs and the like for diseases related to the urinaryorgan/reproductive organ system can be expected from the medicamentwhich contains, as the active ingredient, a compound, peptide orantibody capable of specifically modifying their activities. Inaddition, since a member of the G protein-coupled receptors of theinvention, such as SREB1 protein, is expressed not only in the centralnervous system and urinary organ/reproductive organ system but also inthe heart and peripheral leukocytes, a medicament which contains, as theactive ingredient, a compound, peptide or antibody capable ofspecifically modifying the activity of SREB1 protein can be expected forits usefulness as therapeutic drugs and the like for circulatory systemdiseases and immune inflammation system diseases, in addition to centraldiseases and diseases related to the urinary organ/reproductive organsystem.

The novel G protein-coupled receptor family SREB1, SREB2 or SREB3 of theinvention has markedly high conservation ratio of amino acids in humanand rat. This conservation ratio is most highest among the existing Gprotein-coupled receptor families, which seems to show that the novel Gprotein-coupled receptor family of SREB1, SREB2 and SREB3 is takingimportant roles in the living body, particularly a physiological role inthe central nervous system. Also, since their amino acid sequences havea conversation ratio of 97% or more in human and rat, it is consideredthat almost no interspecies differences are present regarding activitiesof drugs which act upon the novel G protein-coupled receptor familySREB1, SREB2 or SREB3. In consequence, when the G protein-coupledreceptor protein of the invention itself or a compound or proteinobtained by a screening using the receptor is developed as a medicament,the receptor has an advantage in that animal experiments using rats, forexample, can be carried out in advance, prior to testing pharmacologicaleffects on human, and is useful in terms that clinical data on human canbe easily predicted from the animal experiment data.

Since expression of the G protein-coupled receptor proteins of theinvention in organs and changes thereof can be detected by the methodsuch as ELISA, radioimmunoassay, the Western blotting and the like usingthe antibodies, these antibodies for the novel G-protein coupledreceptor proteins are useful as diagnostic agents. In addition, theantibodies capable of modifying activities of the novel Gprotein-coupled receptor proteins are useful as therapeutic drugs fordiseases in which the novel G protein-coupled receptor proteins areinvolved and also as tools for the separation and purification of thereceptor proteins.

1. An isolated polynucleotide encoding a G protein-coupled receptorprotein comprising amino acids 1 to 375 of SEQ ID NO:2.
 2. The isolatedpolynucleotide encoding a G protein-coupled receptor protein of claim 1,which encodes a polypeptide consisting of amino acids 1 to 375 of SEQ IDNO:2.
 3. An expression vector comprising the polynucleotide of claim 1or
 2. 4. A host cell transformed or transfected with the expressionvector of claim
 3. 5. A process for producing a recombinant host cellcomprising transforming or transfecting a cell with the expressionvector of claim 3 such that the host cell, under appropriate conditions,produces said G protein-coupled receptor protein.
 6. A membrane of therecombinant host cell of claim 4 expressing said polypeptide.
 7. Amethod for producing a G protein-coupled receptor protein comprisingculturing the host cell of claim 4 under conditions sufficient for theproduction of said G protein-coupled receptor protein and recoveringsaid protein from the culture.
 8. A method for producing a Gprotein-coupled receptor protein which has the amino acid sequencecomprising amino acids 1 to 375 of SEQ ID NO:2 comprising culturing thehost cell of claim 4.